Herpesviridae (herpesviruses or herpes family viruses) is the name of a family of enveloped, double-stranded DNA viruses with relatively large complex genomes. They replicate in the nucleus of a wide range of vertebrate hosts, including eight varieties isolated in humans, several each in horses, cattle, mice, pigs, chickens, turtles, lizards, fish, and even in some invertebrates, such as oysters. Human herpesvirus infections are endemic and sexual contact is a significant method of transmission for several including both herpes simplex virus 1 and 2 (HSV-1, HSV-2), also human cytomegalovirus (HHV-5) and likely Karposi's sarcoma herpesvirus (HHV-8). The increasing prevalence of genital herpes and corresponding rise of neonatal infection and the implication of Epstein-Barr virus (HHV-4) and Karposi's sarcoma herpesvirus as cofactors in human cancers create an urgency for a better understanding of this complex, and highly successful virus family.
The virion structure of all herpesvirus virions are comprised of four structural elements: 1. Core: The core consists of a single linear molecule of dsDNA in the form of a torus. 2. Capsid: Surrounding the core is an icosahedral capsid with a 100 nm diameter constructed of 162 capsomeres. 3. Tegument: Between the capsid and envelope is an amorphous, sometimes asymmetrical, feature named the tegument. It consists of viral enzymes, some of which are needed to take control of the cell's chemical processes and subvert them to virion production, some of which defend against the host cell's immediate responses, and others for which the function is not yet understood. 4. Envelope: The envelope is the outer layer of the virion and is composed of altered host membrane and a dozen unique viral glycoproteins. They appear in electron micrographs as short spikes embedded in the envelope.
The herpesvirus genomes range in length from 120 to 230 kbp with base composition from 31% to 75% G+C content and contain 60 to 120 genes. Because replication takes place inside the nucleus, herpesviruses can use both the host's transcription machinery and DNA repair enzymes to support a large genome with complex arrays of genes. Herpesvirus genes, like the genes of their eukaryotic hosts, are not arranged in operons and in most cases have individual promoters. However, unlike eukaryotic genes, very few herpesvirus genes are spliced.
The genes are characterized as either essential or dispensable for growth in cell culture. Essential genes regulate transcription and are needed to construct the virion. Dispensable genes for the most part function to enhance the cellular environment for virus production, to defend the virus from the host immune system and to promote cell to cell spread. The large numbers of dispensable genes are in reality required for a productive in vivo infection. It is only in the restricted environment of laboratory cell cultures that they are dispensable. All herpesvirus genomes contain lengthy terminal repeats both direct and inverted. There are six terminal repeat arrangements and understanding how these repeats function in viral success is an interesting part of current research.
Four biological properties that characterize members of the herpesviridae family are that herpesviruses express a large number of enzymes involved in metabolism of nucleic acid (e.g. thymidine kinase), DNA synthesis (e.g. DNA helicase/primase) and processing of proteins (e.g. protein kinase); herpesviruses synthesize viral genomes and assemble capsids within the nucleus; their productive viral infection is accompanied by inevitable cell destruction; and herpesviruses are able to establish and maintain a latent state in their host and reactivate following cellular stress. Latency involves stable maintenance of the viral genome in the nucleus with limited expression of a small subset of viral genes.
Herpes virus family, which includes cytomeglavirus and herpes simplex virus, is found in the body fluids of infected individuals including urine, saliva, breast milk, blood, tears, semen, and vaginal fluids.
In the U.S., between 50% and 80% of adults are positive for HCMV by the age of 40 and there is no cure. While most infections are ‘silent’, HCMV can cause disease in unborn babies and immunocompromised people. HCMV in positive mothers can lead to Down syndrome, fetal alcohol syndrome, and neural tube defects. Furthermore, approximately 33% of women who become infected with HCMV for the first time during pregnancy pass the virus to unborn babies. Currently, 1 in 150 babies is born with congenital HCMV infection and 1 in 750 babies is born with or develops permanent disabilities dues to HCMV. Moreover, HCMV is widespread in developing countries and areas of lower socioeconomic conditions. Therefore, developing a preventative and/or therapeutic vaccine against HCMV would decrease morbidity and medical costs associated with virus-associated illness and disease worldwide.
Current vaccine strategies using attenuated/killed virus or recombinant proteins have been reported to yield levels of efficacy approaching 35% at best. Since antibodies (Abs) recognizing viral glycoproteins such as gB, gH, gM, and gN are observed in cases of protection, it is thought that the elicitation of neutralizing Abs against these viral surface targets are important. Furthermore, T cell epitopes are known to occur in particular viral proteins including UL83 (pp 65), which specifically defines T-cell-based vaccine approaches targeting pp65 epitopes.
The direct administration of nucleic acid sequences to vaccinate against animal and human diseases has been studied and much effort has focused on effective and efficient means of nucleic acid delivery in order to yield necessary expression of the desired antigens, resulting immunogenic response and ultimately the success of this technique.
DNA vaccines have many conceptual advantages over more traditional vaccination methods, such as live attenuated viruses and recombinant protein-based vaccines. DNA vaccines are safe, stable, easily produced, and well tolerated in humans with preclinical trials indicating little evidence of plasmid integration [Martin, T., et al., Plasmid DNA malaria vaccine: the potential for genomic integration after intramuscular injection. Hum Gene Ther, 1999. 10(5): p. 759-68; Nichols, W. W., et al., Potential DNA vaccine integration into host cell genome. Ann N Y Acad Sci, 1995. 772: p. 30-9]. In addition, DNA vaccines are well suited for repeated administration due to the fact that efficacy of the vaccine is not influenced by pre-existing antibody titers to the vector [Chattergoon, M., J. Boyer, and D. B. Weiner, Genetic immunization: a new era in vaccines and immune therapeutics. FASEB J, 1997. 11(10): p. 753-63]. However, one major obstacle for the clinical adoption of DNA vaccines has been a decrease in the platform's immunogenicity when moving to larger animals [Liu, M. A. and J. B. Ulmer, Human clinical trials of plasmid DNA vaccines. Adv Genet, 2005. 55: p. 25-40]. Recent technological advances in the engineering of DNA vaccine immunogen, such has codon optimization, RNA optimization and the addition of immunoglobulin leader sequences have improved expression and immunogenicity of DNA vaccines [Andre, S., et al., Increased immune response elicited by DNA vaccination with a synthetic gp120 sequence with optimized codon usage. J Virol, 1998. 72(2): p. 1497-503; Deml, L., et al., Multiple effects of codon usage optimization on expression and immunogenicity of DNA candidate vaccines encoding the human immunodeficiency virus type 1 Gag protein. J Virol, 2001. 75(22): p. 10991-1001; Laddy, D. J., et al., Immunogenicity of novel consensus-based DNA vaccines against avian influenza. Vaccine, 2007. 25(16): p. 2984-9; Frelin, L., et al., Codon optimization and mRNA amplification effectively enhances the immunogenicity of the hepatitis C virus nonstructural 3/4A gene. Gene Ther, 2004. 11(6): p. 522-33], as well as, recently developed technology in plasmid delivery systems such as electroporation [Hirao, L. A., et al., Intradermal/subcutaneous immunization by electroporation improves plasmid vaccine delivery and potency in pigs and rhesus macaques. Vaccine, 2008. 26(3): p. 440-8; Luckay, A., et al., Effect of plasmid DNA vaccine design and in vivo electroporation on the resulting vaccine-specific immune responses in rhesus macaques. J Virol, 2007. 81(10): p. 5257-69; Ahlen, G., et al., In vivo electroporation enhances the immunogenicity of hepatitis C virus nonstructural 3/4A DNA by increased local DNA uptake, protein expression, inflammation, and infiltration of CD3+ T cells. J Immunol, 2007. 179(7): p. 4741-53]. In addition, studies have suggested that the use of consensus immunogens can be able to increase the breadth of the cellular immune response as compared to native antigens alone [Yan, J., et al., Enhanced cellular immune responses elicited by an engineered HIV-1 subtype B consensus-based envelope DNA vaccine. Mol Ther, 2007. 15(2): p. 411-21; Rolland, M., et al., Reconstruction and function of ancestral center-of-tree human immunodeficiency virus type 1 proteins. J Virol, 2007. 81(16): p. 8507-14].
One method for delivering nucleic acid sequences such as plasmid DNA is the electroporation (EP) technique. The technique has been used in human clinical trials to deliver anti-cancer drugs, such as bleomycin, and in many preclinical studies on a large number of animal species.
There remains a need for nucleic acid constructs that encode herpesvirus antigens and for compositions useful to induce immune responses against herpesviruses. There remains a need for effective vaccines against herpesviruses that are economical and effective.